ARO80 Thoughts and Plans

T is half period...yes it's weird, but less confusing than using w all the time

t is the time along the experiment

I is the average nuclear intensity

TO DO

Negative Control (As asked by first referee)

Same timings as normal experiments (e.g. 3hr, 10min imaging), but no oscillations at all and measure end intensity values : WT,GAP1,WAP1

Currently I have a WT trial where +tyr starts at T = 0 and then I record everything

What the first referee wants

Clearly show the role of permeases

So we have shown it quite clearly with GAP1, now onto WAP1

Are they physiologically important

Not sure how exactly we are addressing this other than looking at the bandwidth

What the second referee wants

I find it very odd that cells do not respond at all at high frequencies.

From my results, I get a response at high frequencies. e.g. at T = 60, t = 0, I = ~30; at T = 60, t = 18, I = ~50

In measuring the frequency response of the cells, the authors acquired data at different rates for different experiments. For instance, when the drive period was 1 min, the acquisition period was 30 sec – but when the drive period was 180 min, the acquisition period was 10 min. Taking two different fluorescent images every thirty seconds puts a lot of stress on the cells. It can also photobleach your reporters. Without the proper controls to make sure these effects are not happening I don’t really trust the data. Plus, if you are only going to report the value of the reporter at some arbitrary time after the beginning of the experiment, why do time-lapse at all?

The methods we came up with for my set of experiments have consistent acquisition periods and total lengths. The time-lapse is to ensure that the accumulation is running as expected and also to make sure that we did not overshoot the maximal intensity i.e. it never decreases.

In section 3.1 and 3.2, the number of sample cells varies greatly in each oscillatory condition, from 7 to 111 cells. This can significantly affect the variation of protein accumulation. The number of quantified cells, especially T = 6 min, should be made comparable to each other. In general, this makes me wonder what is really going on in these experiments that the number of cells varies so greatly.

While I don't have exactly consistent number of cells between different frequencies, I do have at least 300 in each so I think this addresses the concern

The authors did not mention whether the experiment in Section 3.1 and 3.2 were replicated. If not, additional experiments are needed for better quantification.

No replicates currently....

Figures 6 and 7 are very confusing, and the discussion of them is extremely hard to follow. It’s not clear to me what these figures add to the study.

Plus, are there any error bars for those plots?

In progress, looking at this stuff now

The ploidy and mating type of the strains used in several experiments is not consistent. For example, in Section 3.5, the control and Gap1p strain is Mat-a, whereas the Wap1p strain is Mat-alpha. Also, while most strains used in the experiments are haploid, the experiment of overexpression of ARO80 used a diploid strain (Fig. S1 legend) - this increaset of gene copy number in the pathway can affect the pathway induction. These points need to be clarified.

The strains I have been using are all consistent: yMM1164(WT), yMM1238(Gap1 - del), and yMM1239(Wap1 - del) are all Mat a HAP1+ARO9-yECitrine-KanMX.

Not sure about this one

In Fig. 3b (T = 1 min), based on microfluidic setup, rhodamine’s signal should follow square-wave function. Thus, a new plot of rhodamine’s signal for this condition is needed.

yup yup have rhodamine at a wide range of periods, all mostly square

In section 3.2., the decay figure of Aro9p (Fig. S3 in the ESI) should be changed to Fig. S5. Also, this decay rate is applied to Aro9-GFP which can be very different to that of Aro9. Thus, the decay rate of Aro9-GFP and PARO9-GFP need to be compared.

TBD

Section 3.3, the authors mentioned one target of Aro80p is ARO8, this should be corrected to ARO80. Also, the authors should explain why an increase in the mRNA level of ARO80 did not lead to increase in expression level of ARO80 protein.

TBD

Annotations of yeast strain are confusing, especially in the strain construction section (supplemental information). Listing genotype of every strain, follow by its codename, in the text made it very difficult to follow the details. Strains’ annotations need to be rewritten. Plus, it seems like the total number of strains is disproportionate to the experimental results reported.